The inner ear is essential for human health as it provides key sensory information required for hearing and balance control. The auditory and vestibular components of the inner ear are fluid-filled chambers that function to detect sound waves and gravity/acceleration/movement, respectively. A greater understanding of the molecules which regulate the production of the endolymph fluid in the inner ear should provide insight into hearing and balance disorders that affect the human population. Preliminary studies show that adult mice mutant for the EphB2 receptor tyrosine kinase display a circling locomotion consistent with a defect in vestibular function. In related preliminary investigations, adult mice heterozygous for a mutation in its cognate transmembrane partner, ephrin-B2, are also found to exhibit a circling locomotion. Both mutants display a much reduced production of endolymph fluid in the vestibular apparatus, which is normally rich in potassium. Direct measurement of the extracellular fluid in the utricle reveals a highly significant decrease in endolymph potassium concentration and endolymphatic potential in the EphB2 and ephrin-B2 mutants. Such a defect in fluid production is consistent with the restricted expression of EphB2 to the secretory vestibular dark cells and ephrin-B2 to adjacent vestibular transitional cells. Dark and transitional cells are subcompartments of the membranous inner ear epithelia that play important roles in regulating the ionic homeostasis of endolymph fluid. As the ephrins and Eph receptors are thought to play important roles in cell-cell signaling, the preliminary data described in this application provides novel insight into the signaling molecules that may control the production and homeostasis of endolymph fluid. Importantly, the data provide two new animal model systems to study vestibular dysfunction. We plan to expand on these genetic experiments in Specific Aims 1 and 2 by generating point mutations in the EphB2 and ephrin-B2 genes to help determine whether tyrosine kinase or PDZ signaling pathways are utilized for endolymph production. Additional preliminary biochemical studies indicate certain PDZ domain proteins that bind the C-terminal tails of both EphB2 and ephrin-B2 are also able to bind the C-terminal tails of other membrane spanning proteins implicated in regulating fluid homeostasis, including aquaporin water channels and anion exchangers. In Specific Aim 3, we plan to expand on these biochemical studies by focusing on possible physical associations of EphB2 with molecules implicated to have important roles in maintaining endolymph homeostasis. The overall objective of the genetic and biochemical studies outlined here are to obtain a better understanding how signaling mediated by EphB2 and ephrin-B2 is linked to the production of endolymph fluid and normal vestibular function.